The Importance of a Broad Consortium of Bacteria for a Healthy Gastrointestinal tract: A Narrative Review of the Live Biotherapeutic Product REBYOTA

Introduction The human microbiome comprises an estimated 100 trillion organisms and their genetic content which consists of bacteria, viruses, fungi, and parasites. There are more than 1,000 different species of bacteria in a healthy gut with the majority residing in the lumen of the large intestine. The balance of these organisms is vital for the balance of health and the relative abundance is essential to provide the various metabolites and other chemicals necessary for normal microbial environment which is known as eubiosis. When the balance or relative abundance is shifted, a disturbance of metabolites can lead to a condition, dysbiosis. Certain organisms are responsible for essential metabolites such as short chain fatty acids or bile acids. These molecules have various consequences including disruption of colonization resistance or lack of GABA production which has been shown to affect the gut-brain axis. It is the relative amounts of certain taxa or species that ensure this balance among these various organisms constitute a broad consortium. This consortium comprises Bacteroidia and Firmicutes which includes Clostridium species. Herein we describe the important features of various organisms and their metabolites. diverse niches, but also from the many ways in which these species interact. The array of taxa identified in various niches is considerable with the most species identified in the gut while microbiomes have been identified in every other niche from the placenta, skin, lung, mouth and even the brain. Often these microbiomes evolve over time. As new ‘omic’ methods are developed, our understanding of the


Introduction
e human microbiome comprises an estimated 100 trillion organisms and their genetic content which consists of bacteria, viruses, fungi, and parasites.ere are more than 1,000 di erent species of bacteria in a healthy gut with the majority residing in the lumen of the large intestine.e balance of these organisms is vital for the balance of health and the relative abundance is essential to provide the various metabolites and other chemicals necessary for normal microbial environment which is known as eubiosis.When the balance or relative abundance is shi ed, a disturbance of metabolites can lead to a condition, dysbiosis.Certain organisms are responsible for essential metabolites such as short chain fatty acids or bile acids.ese molecules have various consequences including disruption of colonization resistance or lack of GABA production which has been shown to a ect the gut-brain axis.It is the relative amounts of certain taxa or species that ensure this balance among these various organisms constitute a broad consortium.is consortium comprises Bacteroidia and Firmicutes which includes Clostridium species.Herein we describe the important features of various organisms and their metabolites.diverse niches, but also from the many ways in which these species interact.e array of taxa identi ed in various niches is considerable with the most species identi ed in the gut while microbiomes have been identi ed in every other niche from the placenta, skin, lung, mouth and even the brain.O en these microbiomes evolve over time.As new 'omic' methods are developed, our understanding of the changes in these niches will improve our ability to identify key organisms in the healthy environment [2].e term 'microbiota' refers to the roughly 1100 species of bacteria, archaea, microeukaryotes, and viruses that share the human body space, and function in commensal, symbiotic, or pathogenic relationships [3,4].e dominant bacterial phyla in the gastrointestinal (GI) tract include Bacteroidetes (or Bacteroidota) and Firmicutes, followed by Actinobacteria and Proteobacteria [5][6][7].
It is important to note that the various microbial taxa do not exert equivalent impacts on the GI ecosystem.
e GI tract can be considered a functionally redundant space whereby the functioning of the system may remain una ected by the loss of some species due to compensation by other similarly functioning organisms.is resilience and stability of the gut microbiome is essential to human health and development.
In contrast, foundational and keystone species in the GI tract have unique and in uential roles [8]: Darwin summarized the complexity of the living world in "On the Origin of Species".He hypothesized about a "tangled bank", inhabited by a multitude of species of plants, birds, insects, and worms, all "dependent upon each other in so complex a manner" [1].Only now are we beginning to explore these same complexities in the microbial world.Like Darwin's tangled bank, microbial ecosystems include not only the remarkably high number of species inhabiting • A foundational species is one that dictates the structure of the ecosystem by creating stable conditions for other species, and by modulating and stabilizing fundamental processes.
• A keystone species is vital for maintaining the organization and diversity of microbial consortium via biotic and abiotic interactions with community members.ese o en modestly Sphingolipids are membrane components and important structural as well as signaling molecules that regulate inflammation and immunity.A variety of Bacteroidetes-derived sphingolipids including ceramide phosphoinositol and deoxy-sphingolipids have been identified.These sphingolipids are critical for maintaining homeostasis and symbiosis in the gut [18].
Gut microbes including Bacteroidia can produce metabolites with high neuroactive potential (neurotransmitters), including γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain.Multiple Bacteroides sp strains produce GABA through a highly conserved glutamate decarboxylase (GAD)-system composed of a GAD, a glutaminase, a glutamate/GABA antiporter, and a potassium channel.All four genes of the GAD-system are present in 90% of Bacteroides genomes.B. ovatus synthesizes GABA and modulates the levels of tryptophan and glutamine [19,20].
The type VI secretion system (T6SS) The type VI secretion system (T6SS) is a molecular conduit that enables Gram-negative bacteria the ability to translocate effector proteins directly into neighboring cells.The T6SS in Bacteroidetes provides an important contribution to colonization resistance and commensal stability [21].
Bacteroidetocins are a family of broad-spectrum peptide toxins produced by Bacteroidetes species that have inhibitory activity against diverse Bacteroides and Parabacteroides species, including environmental, symbiotic, and pathogenic members [22].
Bacteroides uniformis encodes structurally and functionally distinct Β-glucuronidases with specific roles in processing drug-glucuronide and glycan substrates, highlighting the broad structural and functional diversity among these enzymes within a single human gut microbe [23].
Bacteroides vulgatus FTJS7K1 was found to protect against lipopolysaccharide-induced acute intestinal injury via modulation of cytokine production in the colon tissue and regulation of the structure of the gut microbiota [24].
Bacteroides thetaiotaomicron administration has been found to inhibit the colonization of Clostridioides difficile and inhibit inflammation in the colon by attenuating lymphocyte infiltration in the mucous layer and decreasing edema in the submucosa [25].
The polysaccharide capsule of B. thetaiotaomicron plays an important role in colonization resistance, suppression of toxin release and production of B-lactamases which can maintain the balanced microbiota [26][27][28].

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The spore-forming clusters IV and XIVa (also known as Clostridium leptum and coccoides groups, respectively) control CD4 + T regulatory cells (Tregs), which play a critical role in the maintenance of immune homeostasis [30].
C. scindens, C. scindens, a member of the Clostridium XlVa clade, has also been shown to produce butyrate, which plays a role in colitis resolution, and secretes the tryptophan-derived antibiotic, 1-acetyl-β-carboline that inhibits multiple gram-positive pathogens found in the gut, including Clostridioides difficile [31,32].
Firmicutes exhibited a positive correlation with serum total testosterone levels independent of host factors (e.g., age, body mass index, triglyceride, and total cholesterol).Therefore, Firmicutes may play a role in testosterone metabolism [33].

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Akkermansia muciniphila is a gram-negative anaerobe that decreased serum aspartate transaminase and alanine transaminase, alleviated liver histopathological damage, substantially attenuated serum levels of pro-inflammatory cytokines and chemokines, significantly decreased hepatocellular apoptosis, and strengthened intestinal barriers and reduced systemic LPS level.A. muciniphila also increased the diversity and richness of the microbial community and simultaneously modulated the varieties of gut microbes, such Lachnospiraceae and Ruminococcaceae [34].Trosvik and Mulnick (2015) used multiple genomic approaches to demonstrate connectedness in the ecological network as a proxy for a taxon's importance in ecosystem dynamics.Actinobacteria t the description of a "keystone taxon" due to its high degree of ecological connectedness and positive in uence within the community despite its low relative abundance [8].
While the above organisms are involved in interactions that stabilize the ecosystem via metabolic, immunologic, and physiologic impacts, they are not the only bene cial species.A complex and diverse microbiota also includes Bi dobacteria, Prevotella, Akkermansia, and members of Firmicutes such as the Ruminococcaceae, Lachnospiracaea, and Clostridiales [9,10].
erefore, a broad consortium of bacterial phyla is re ective of a healthy, complex microbiome.Indeed, these communities in aggregate may be referred to as a metabolic organ with crucial functions that exert wide-ranging impacts on human health.e foundation taxon, Bacteroidetes, has a very broad metabolic and physiologic potential that includes immunomodulatory e ectsas well as the ability to adapt and persist in changing gut environments, thus contributing to the stability of the microbiome [8,11,12].Firmicutes, the most abundant and diverse gut species [13] has functions that include anti-in ammatory e ects [14,15] and forti cation of the gut barrier [16].Both of these phyla are key producers of short chain fatty acids (SCFAs), an important energy source for host intestinal cells that constitute the gut barrier and abundant species underscore vulnerabilities in the homeostasis of the ecosystem since their abrogation can result in a loss of biodiversity as well as key functions

Live biotherapeutic products Clostridioides difficile infection (CDI)
Identi cation of the GI tract organisms that potentiate gut health and provide colonization resistance against pathogens underpins emerging and novel therapies such as precision fecal microbiome transplantation (FMT).Currently, the consortium containing only spore-forming bacteria, VOWST (formerly SER-109), Seres erapeutics) was FDA approved in April 2023 and the other select consortium, Vedanta candidate VE303 is in Phase III clinical trials.e broad consortium human derived product, REBYOTA (RBL; Ferring Pharmaceuticals), was recently approved by the FDA.Uniquely RBL contains a wide range of bacterial species (10 8 -10 10 organisms per dose) including >1x10 5 colony forming units (CFU) per mL of Bacteroides spp.Other organisms include members of the Firmicutes, Faecalibacteria, Roseburia, Blautia, and other non-spore-forming bacteria [67].Recent data published from the large clinical program on RBL demonstrated good safety and e cacy along with replenishment of a healthy state of the diseased dysbiotic bowel associated with rCDI [68].About 30% of patients had a rst recurrence post index infection [69] and importantly, two studies followed patients for two years and no cases of bacteremia or fungemia were reported [70,71].Additionally the single dose group achieved a sustained clinical response of 95.7% over two years [72].
ere is a totality of evidence from RBL-or placebo-treated participants in the randomized, double-blind, placebo-controlled trials PUNCH CD2 (NCT02589847; n = 153) and PUNCH CD3 (NCT03244644; n = 289, and from RBL-treated participants in the PUNCH OLS (NCT03931941; n = 653) that clinical response to RBL is associated with restoration of microbiome (Figure 2).Furthermore, clinical response to RBL is signi cantly associated with engra ment (Figure 3); the phylum Bacteroidia and the Firmicute phylum including the genus Clostridia showed the highest engra ment percentages in RBL-treated participants wherein 16/30 e ective engra ers were Bacteroidia and nine were Clostridia.Speci cally, 58% of B. uniformis species were characterized as e ective engra ers [73].
Clostridioides di cile infection (CDI) is the most common cause of antibiotic-and healthcare-associated infectious diarrhea in the United States [35,36], associated with almost half a million infections and 29,300 deaths annually [35,37].CDI imposes substantial clinical, psychological, social, and professional burdens [38], with some studies estimating its attributing inpatient treatment and mitigation costs to be as high as $5 billion annually [39].e causative agent, C. di cile, is a fastidiously anaerobic, Gram-positive, spore-forming bacterium; toxin-producing (toxigenic) strains can produce up to three exotoxins, Toxin A, Toxin B, and binary toxin [40].e incidence of recurrent CDI (rCDI) has also increased signicantly in recent years, and this has been identi ed as a major public health challenge [35,[41][42][43][44]. Especially in the elderly, up to 35% of patients who initially respond to antimicrobial therapy experience disease recurrence, of which 60% experience subsequent recurrences [45].
Bacteroides thetaiotaomicron attenuates colonization of C. di cile and in ammation in the colon.It suppresses toxin release from C. di cile by inhibiting cell autolysis [27] and helps restore healthy microbial balance by increasing Bacteroidetes and lowering Proteobacteria levels [25].
Bacteroides fragilis prevents CDI by restoring the gut barrier (by eliciting robust ZO-1 and Muc-2 expression), inhibiting C. di cile adherence, attenuating the decrease in CDI-induced transepithelial electrical resistance, and improving commensal bacterial diversity and relative abundance [65].Finally, members of the Lachnospiraceae and Ruminococcaceae families of Clostridiales order may harbor coprostanoligenic activity; this cholesterol-reducing e ect suggests that these particular bacteria may play an important role not only in host lipid metabolism, but also susceptibility to CDI [66].
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secrete vital immunomodulatory factors.Multiple species in the Bacteroidetes phylum produce propionate and acetate, while Firmicutes produce propionate and butyrate [17].Figure 1 illustrates examples of relevant functions of these keystone species in maintaining intestinal homeostasis.
In essence, a "healthy" microbiota requires a diverse consortium of microbial organisms, including both Firmicutes and Bacteroidetes and other key organisms that in concert support vital functions of the intestinal tract.e speci c microbial composition likely di ers from individual to individual as this is in uenced by various host factors such as diet, lifestyle, comorbidities, medications, and others.Systemic or local conditions that skew the distribution or functioning of the microbial community (or a state of dysbiosis) may increase susceptibility to certain diseases.

Summary key points
• e ecology of the healthy human Gl tract is dictated by a broad consortium of microorganisms including Bacteroidia as well as Firmicutes such as Clostridium species.
• Bacteroidetes modulate bile acid and SCFA metabolism to control C. di cile colonization, spore germination, and persistence.
• Bacteroidetes also exert other important functions including host immunity modulation, gut wall integrity, and general colonization-resistance.
• Firmicutes provide key metabolic pathways, such as SCFA metabolism, that augment the activities of Bacteroidetes.
• e broad consortium of microbiota is therefore a "metabolic organ" underpinning diverse aspects of human GI tract health.
• RBL is a broad consortium human derived product that demonstrates safety and e cacy up to two years with positive shi s in microbiota.

6.
Longer bars mean higher engraftment

Figure 1
Figure 1 Specific functions of Bacteroidetes and Firmicutes

Figure 2 Figure 3
Figure 2Clinical response to RBL is associated with a microbiota shift reminiscent of a healthy gut consortium[73]